A simple and sensitive assay of total serum bile acids

Abstract

A simple and sensitive assay system was presented for the determination of total bile acids in serum. One tenth ml of serum in tube (A) and the same amount of distilled water in tube (C) were added with Tris-aminomethane hydrochloric buffer and inactivated at 67° for 20 min. After heating 3α-hydroxysteroid: NAD oxydoreductase (EC. 1.1.1.50, 3α-HSD), NAD, diaphorase (EC. 1.6.4.3.), and resazurin were added to this solution. This mixture was incubated at 20° for 40 min. As blank another 0.1ml of the same serum in tube (B) and the same amount of distilled water in tube (D) were made after the same treatment in the same assay system without 3α-HSD as tubes (A) and (C). The fluorescence of resorfin which was converted from resazurin in these tubes were measured at 580 nm with the excitation at 560nm. ΔF was calculated as follows and the amount of bile acids was obtained according to the standard curve. ΔF=fluorescence intensity in tube (A)-(C)-[(B)-(D)] Relationship between the amount of standard bile acid and the fluorescence intensity was linear in the range of 0 to 50uM.
About ninety percent of recovery was gained irrespective of the species and forms of conjugation of bile acids when added to serum. Reproducibility in a serum was 6.5uM with standard deviation of±0.20 and C. V. of 3.1%.
Normal value in fasting serum was found in the range of 4.5uM to 9.2uM with mean of 6.4uM in male (n=9) and 3.8 to 6.5uM with mean of 4.8uM in female (n=5). Constant values were obtained in sera of a female which were taken successively for five days in the fasting state.
Serum bile acids level was elevated after eating egg yolk in all eight normal subjects tested.
Described method is an improved one which was reported in detail in “Clinica Chimica Acta” (in press, 1976)